Compositions for in situ nucleic acid analysis

ABSTRACT

Sample preparation processes for in situ RNA or DNA analysis, methods and compositions therefor are provided. Processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or extraction. The preparation process can be carried out at room temperature in as little as seven minutes and is amenable to high throughput processing using manual or robotic platforms.

The present application is a continuation of pending U.S. applicationSer. No. 14/459,805 filed Aug. 14, 2014, now U.S. Pat. No. 9,279,152,Mar. 8, 2016, which application is a divisional of U.S. application Ser.No. 14/136,685 filed Dec. 20, 2013 (now U.S. Pat. No. 8,828,664 whichgranted Sep. 9, 2014), which application is a divisional of U.S.application Ser. No. 13/650,853 filed Oct. 12, 2012 (now abandoned),which application is a continuation of U.S. application Ser. No.13/157,840 filed Jun. 10, 2011 (now U.S. Pat. No. 8,288,106 whichgranted Oct. 16, 2012), which application is a divisional application ofU.S. application Ser. No. 12/122,274 filed May 16, 2008 (now U.S. Pat.No. 7,964,350 which granted Jun. 21, 2011), which application claims thebenefit of U.S. Provisional Application No. 60/938,978 filed May 18,2007. The contents of all applications are incorporated herein in theirentirety.

The U.S. government has certain rights in this application pursuant toGrant No. R44HL69718 from the National Institutes of Health.

FIELD

The present teachings generally relate to compositions, processes,methods, and kits for preparation of samples containing genetic materialfor analysis, detection and/or quantitation.

INTRODUCTION

Real-time polymerase chain reaction (PCR) is routinely used fordetection of nucleic acid, and real-time quantitative reversetranscriptase-PCR (qRT-PCR) is routinely used for detection of RNA andfor studying gene expression. However, current procedures for carryingout RT-PCR directly on cell lysates are not amendable to high throughputanalysis using robotic workstations. For example, procedures forpreparation of nucleic acid for detection requiring temperature shiftsmake controlling temperature across an entire plate or block achallenge. Certain current procedures also contain components that areinhibitory for optimum reverse transcriptase function or for optimum DNApolymerase function. The present teachings provide composition, methodand kit embodiments for preparation of samples for detection and/orquantitation of nucleic acid that are amenable to high throughputanalyses.

SUMMARY

Certain trademarked products are cited by teachings herein withreference to surfactants. Generic descriptions for such products are asfollows: TRITON X-100™, octylphenol ethoxylate having an average of 9.5ethoxylate groups (Dow Chemical Company Product Information, Form No.119-01882, JMS1206); TRITON X-114™, octylphenol ethoxylate having anaverage of 7.5 ethoxylate groups (Dow Chemical Company ProductInformation, Form No. 119-01884, JMS 1206); NONIDET P-40™,octylphenolpoly(ethyleneglycolether) (Roche Diagnostics GmbH, CatalogNo. 11 332 473 001, July 2005); and THESIT™, dodecyl alcoholpolyoxyethylene ether (IUPAC Name 2-dodecoxyethanol; CAS Number9002-92-0; Chemical Formula C₁₄H₃₀O₂).

Sample preparation process embodiments provided by teachings hereininclude a process for preparing a sample containing RNA for in situanalysis of RNA or a surrogate thereof. In some embodiments, the processcomprises contacting the sample containing RNA with a lysis mixtureunder conditions and for a time to produce a lysate, and admixing thelysate with a stop mixture at substantially the same temperature as thecontacting step to form a stopped mixture. For such embodiments, thelysis mixture comprises a polypeptide having protease activity, apolypeptide having deoxyribonuclease activity, and a surfactant thatsubstantially lacks fluorescence between 300 nm and 750 nm when in usefor in situ analysis of RNA or a surrogate thereof. Also, for suchembodiments, the lysis mixture is substantially free of a cationchelator. The stop mixture comprises a cation chelator effective toinactivate the polypeptide having deoxyribonuclease activity, and aninhibitor of the polypeptide having protease activity. The resultantstopped mixture is compatible with in situ reverse transcriptase and DNApolymerase reactions. In some embodiments, the stop mixture furthercomprises a peptide or molecule having ribonuclease inhibitory activity.

In certain embodiments, the stopped mixture can be further combined withreagents for reverse transcription to form a first amplification mixtureand, in some embodiments, the first amplification mixture is placed incontact with reagents for quantitative polymerase chain reaction (qPCR)amplification. In some embodiments, reagents for qPCR amplificationcomprise a green, yellow or orange emitter, and the process furthercomprises carrying out in situ analysis of the DNA, RNA, or a surrogatethereof comprising detecting fluorescence of the green, yellow, ororange emitter, respectively.

For certain embodiments, the sample preparation process of contactingand admixing are carried out at substantially the same temperature,which temperature is from 15° C. to 30° C., 16° C. to 28° C. or 19° C.to 25° C. as further described infra.

In some embodiments, a process for preparing a sample containing RNA forin situ analysis of RNA or a surrogate thereof is provided, whichprocess comprises contacting the sample containing RNA with a lysismixture at 16° C. to 28° C. for a time to produce a lysate, and admixingthe lysate with a stop mixture at substantially the same temperature asthe contacting step to form a stopped mixture. For such embodiments, thelysis mixture comprises proteinase K or an enzymatically active mutantor variant thereof, DNase I, and a surfactant comprising TRITON X-114™at a concentration from 0.02% to 3%, or 0.05% to 2%, or 0.05% to 1%,THESIT™ at a concentration of 0.01% to 5%, or 0.02% to 3%, or 0.05% to2%, or 0.05% to 1%, or 0.05% to 0.5%, or 0.05% to 0.3%, TRITON X-100™ ata concentration of 0.05% to 3%, or 0.05% to 1%, or 0.05% to 0.3%,NONIDET P-40™ at a concentration of 0.05% to 5%, or 0.1% to 3%, or 0.1%to 2%, or 0.1% to 1% or 0.1% to 0.3% or 0.1% to 5%, or a combinationthereof, and wherein the lysis mixture is substantially free of a cationchelator. Also for such embodiments, the stop mixture comprises a cationchelator in an amount effective to inactivate DNase I, and amethoxysuccinyl-Ala-Ala-Pro-Val-haloalkyl ketone (AAPV, SEQ ID NO:1)wherein the halo is chloro, bromo, iodo, or fluoro and the alkyl isC₁-C₃, such as methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone(AAPV, SEQ ID NO:1) or an active mutant or analog thereof. In someembodiments, the lysis mixture further comprises a calcium salt, areducing agent, or a combination thereof. In some embodiments, thecalcium chelator of the stop mixture comprises ethylene glycoltetraacetic acid (EGTA). In further embodiments, the stop mixturecomprises a ribonuclease inhibitor.

Sample preparation processes for samples containing DNA for in situanalysis of DNA or a surrogate thereof are provided by other embodimentsherein. Such process embodiments comprise contacting the samplecontaining DNA with a lysis mixture at 16° C. to 28° C. for a time andunder conditions to produce a lysate, and admixing the lysate with astop mixture at substantially the same temperature as the contactingstep. For such embodiments, the lysis mixture comprises a polypeptidehaving protease activity, and a surfactant comprising TRITON X-114™ at aconcentration from 0.02% to 3%, or 0.05% to 2%, or 0.05% to 1%, THESIT™at a concentration of 0.01% to 5%, or 0.02% to 3%, or 0.05% to 2%, or0.05% to 1%, or 0.05% to 0.5%, or 0.05% to 0.3%, TRITON X-100™ at aconcentration of 0.05% to 3%, or 0.05% to 1%, or 0.05% to 0.3%, NONIDETP-40™ at a concentration in the lysis mixture of 0.05% to 5%, or 0.1% to3%, or 0.1% to 2%, or 0.1% to 1% or 0.1% to 0.3%, or 0.1%-5%, or acombination thereof. In further embodiments, the lysis mixture comprisesa peptide with ribonuclease activity. In addition, embodiments of thestop mixture comprise an inhibitor of the polypeptide having proteaseactivity. Such an inhibitor has little to no inhibitory activity on DNApolymerase activity.

In further embodiments of teachings herein, a composition for cell lysiscomprises proteinase K, DNase I, and a surfactant comprising TRITONX-114™ at a concentration of 0.05% to 1%, THESIT™ at a concentration of0.05% to 0.3%, TRITON X-100™ at a concentration of 0.05% to 0.3%,NONIDET P-40™ at a concentration of 0.1% to 0.3%, or a combinationthereof, wherein the composition is substantially free of a cationchelator. Embodiments of the composition for cell lysis can furthercomprise a calcium salt.

In some embodiments provided herein, a stop mixture is provided as acomposition of matter, which composition comprises a cation chelator, amethoxysuccinyl-AAPV-haloalkyl ketone (SEQ ID NO:1) wherein the halo ischloro, bromo, iodo, or fluoro and the alkyl is C₁-C₃, and an inhibitorof a ribonuclease.

For certain embodiments, kits for preparation of a sample containing RNAfor in situ detection of RNA or a surrogate thereof are provided whereinthe kits comprise lysis solution components comprising a polypeptidehaving protease activity, and a surfactant comprising TRITON X-114™,THESIT™, TRITON X-100™, NONIDET P-40™, or a combination thereof, apolypeptide having deoxyribonuclease activity, wherein the lysissolution components are substantially free of a cation chelator; andstop mixture components comprising a cation chelator, an inhibitor ofthe polypeptide having protease activity, and, optionally, aribonuclease inhibitor and a reducing agent.

Kit embodiments can further comprise one or more reagents for reversetranscription, such as reverse transcriptase, a reverse primer, dNTPs ora reverse transcriptase buffer, or can further comprise one or morereagents for PCR, such as a DNA polymerase, for example.

In some embodiments, processes and compositions are compatible withdownstream nucleic acid detection methods using methods such as reversetranscription, polymerase chain reaction, qPCR, qRT-PCR, melt curveanalysis, sequencing, message amplification, preamplification,detection, linear amplification for array analysis, and others that useCYANINE™ 3 or CYANINE™ 5 in array analysis, for example. As an exampleof compatibility, results for detection of miR-21 using samplepreparation embodiments described herein were similar to results fromusing the MIRVANA™ kit for isolation of miRNA.

Sample preparation processes provided by embodiments herein are usefulfor any method where RNA or DNA is analyzed, e.g., detected orquantitated. Stopped samples may be used for genotyping analysis, geneexpression analysis, copy number analysis, DNA methylation analysis, SNPgenotyping, plant cell genotyping, or RNA analysis including, forexample, analysis, detection or quantitation of mRNA and noncoding RNAsuch as, for example, rRNA, siRNA, snRNA, or miRNA.

Sample preparation embodiments presented by teachings herein providesurprisingly fast, efficient, and ambient temperature production of alysate that is RT and PCR ready due, in part, to provision of conditionsunder which a protease and a deoxyribonuclease can carry out enzymaticactivity at the same time and in the same reaction mixture. Samplepreparation embodiments presented herein can be performed on cells thatare in suspension or on cells that are attached to a growth surface suchas for 96- or 384-well culture plates. These and other features of thepresent teachings will become more apparent from the description herein.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A provides a schematic diagram depicting a general overview ofcertain exemplary embodiments of the teachings herein for preparation ofnucleic acid for analysis.

FIG. 1B provides a schematic diagram depicting a general overview oftypical existing cell lysate based sample preparation methods involvingmultiple temperature steps.

FIG. 2 provides data demonstrating that 1 mM EDTA appears to have aninhibitory effect on DNase I activity using the DNaseALERT™ assay.DNaseALERT™ substrate fluorescence of Buffer A with EDTA (♦, diamonds),Buffer A without EDTA (●, circles), and DNase I buffer (▪, squares). Thecontrols without DNase I are shown using empty diamonds, circles andsquares and are clustered at the baseline.

FIG. 3 provides data on cell lysis using TRITON X-114™ surfactant at sixdifferent concentrations and using TRITON X-100™ surfactant at oneconcentration as determined by propidium iodide (PI) staining (1 mg/ml)of 10⁵ HeLa cells. Shown are fluorescence intensities of mixturescontaining buffer and PI (shaded bars), buffer and cells (cross-hatchedbars (all are at the baseline)), and buffer and cells and PI (emptybars).

FIG. 4 provides data on the concentration of four separate surfactantsversus cycle threshold using PCR and PPIA (peptidylprolyl isomerase A)as described in Example 2. Data are provided for TRITON X-114™(triangles, solid line), THESIT™ (diamonds, dashed line), TRITON X-100™(squares, dotted line), and NONIDET P-40™ (circles, solid line)surfactants.

FIG. 5 provides data on 5-carboxyfluorescein (5-FAM™) backgroundfluorescence at different concentrations for four different surfactants.Data are provided for TRITON X-114™ (triangles, solid line), THESIT™(diamonds, dashed line), TRITON X-100™ (squares, dotted line), andNONIDET P-40™ (circles, solid line) surfactants.

FIG. 6A-FIG. 6D provide amplification plots of Rn (fluorescencecorrected to a reference ROX™ dye) vs cycle number in the presence ofTHESIT™ (FIG. 6A), TRITON X-100™ (FIG. 6B), TRITON X-114™ (FIG. 6C) andNONIDET P-40™ (FIG. 6D). Concentrations of surfactants are as for FIG. 4and FIG. 5.

FIG. 7 provides an analysis of proteinase K inhibition bymethoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPV, SEQ ID NO:1)for isothermal preparation of samples for processing of nucleic acid.The lower marker is at about 3.5 kDa; the next higher prominent bandsare system peaks at about 4.0-4.5 kDa; intact RNase A is at about 20kDa; and proteinase K is at about 35 kDa. Lanes are labeled as follows:(L) Ladder, (1) RNase Only, (2) PK Only, (3)-(8) AAPV (SEQ ID NO:1) at 1mM (3), 0.75 mM (4), 0.5 mM (5), 0.25 mM (6), 0.125 mM (7), and 0 mM(8).

FIG. 8 provides data showing results from sample processing of HeLacells (10-10⁵ cells per lysis reaction) and analysis using a TAQMAN®Gene Expression Assay for β-actin. There is good linearity down to aninput of as few as 10 cells. (Slope=−3.41, R²=1).

FIG. 9A-FIG. 9B demonstrate that C_(T) values obtained using lysates asprepared using processes provided herein were found to be essentiallyequivalent to C_(T) values obtained with purified RNA.

FIG. 10 provides data demonstrating the reproducibility of samplesprepared according to embodiments herein as compared to purified RNA. 24replicates of 5000 HeLa cells were either subjected to traditional RNApurification or cell lysis using isothermal preparation methods ofteachings herein. Samples were then evaluated using a TAQMAN® GeneExpression Assay for β-actin.

FIG. 11 provides data for detection of SNPs using sample preparationprocesses of embodiments herein as compared to using purified DNA fromthe same cell sources. The data are clustered for cell lines with alleleY at “A,” for cell lines with allele X at “C” and for cell linescontaining both alleles at “B” thereby showing consistency of resultsfrom the two methods. The “D” data provide the no template controlsamples.

DESCRIPTION OF VARIOUS EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. The use of “comprise”, “contain”, and“include”, or modifications of those root words, for example but notlimited to, “comprises”, “contained”, and “including”, are not intendedto be limiting. Use of “or” means “and/or” unless stated otherwise. Theterm “and/or” means that the terms before and after can be takentogether or separately. For illustration purposes, but not as alimitation, “X and/or Y” can mean “X” or “Y” or “X and Y.” As usedherein and unless otherwise indicated, the terms “a” and “an” are takento mean “one,” “at least one” or “one or more.”

Whenever a range of values is provided herein, the range is meant toinclude the starting value and the ending value and a value or valuerange there between unless otherwise specifically stated. For example,“from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35,0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39;and the like.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. All literature and similar materials cited in this applicationincluding, but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines oruses a term in such a way that it contradicts that term's definition inthis application, this application controls. While the present teachingsare described in conjunction with various embodiments, it is notintended that the present teachings be limited to such embodiments. Onthe contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Temperature: The sample preparation processes of teachings hereininclude a contacting step to produce a lysate and an admixing step wherethe lysate is mixed with a stop mixture where the steps are carried outat substantially the same temperature. “Substantially the sametemperature” generally refers to an isothermal process of holding thetemperature relatively constant during the contacting and admixing stepsand, for certain embodiments described herein, means ambient temperaturewhich temperature may change during the day or from lab to lab. Ingeneral, the contacting and admixing steps are carried out atsubstantially the same temperature, which temperature is about 15° C. to40° C., or about 16° C. to 28° C. or about 19° C. to 26° C., or about19° C. to 25° C., or about 22° C. to 25° C., or at ambient temperature,or about 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C.,23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C.,32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40°C. An isothermal process is particularly amenable for high throughputanalyses.

Sample: The term “sample,” as used herein, refers to an in vitro cell,cell culture, virus, bacterial cell, fungal cell, plant cell, bodilysample, or tissue sample that contains genetic material. In certainembodiments, the genetic material of the sample comprises RNA. In otherembodiments, the genetic material of the sample is DNA, or both RNA andDNA. In certain embodiments, a tissue sample includes a cell isolatedfrom a subject. A subject includes any organism from which a sample canbe isolated. Non-limiting examples of organisms include prokaryotes,eukaryotes or archaebacteria, including bacteria, fungi, animals,plants, or protists. The animal, for example, can be a mammal or anon-mammal. The mammal can be, for example, a rabbit, dog, pig, cow,horse, human, or a rodent such as a mouse or rat. In particular aspects,the tissue sample is a human tissue sample. The tissue sample can be,for example, a blood sample. The blood sample can be whole blood or ablood product (e.g., red blood cells, white blood cells, platelets,plasma, serum). The sample, in other non-limiting embodiments, can besaliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissueprint, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinalfluid, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor,synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonaryeffusion, serosal fluid, organs, bronchio-alveolar lavage, tumors,frozen cells, or constituents or components of in vitro cell cultures.In other aspects, the tissue sample is a solid tissue sample or a frozentissue sample. In still further aspects, the sample comprises a virus,bacteria, or fungus. The sample can be an ex vivo tissue or sample or asample obtained by laser capture microdissection. The sample can be afixed sample, including as set forth by U.S. Published PatentApplication No. 2003/0170617 filed Jan. 28, 2003.

Sample preparation processes provided by teachings herein are for fromone cell up to about 10⁵-10⁶ cells per sample or any range therebetween.For certain cell lines, such as HeLa cells, linear C_(T) values wereobtained for up to 10⁶ cells per sample preparation. A patient needlebiopsy often consists of thousands of cells. A biopsy could be preparedusing methods herein, PCR amplified and analyzed by measuring theexpression of certain genes, for example.

In some embodiments, the sample is removed from serum components priorto preparation. In some embodiments, the sample is washed with asolution comprising, for example, but not limited to, phosphate-bufferedsaline (PBS), physiological saline, serum-free media or suitablesolution with appropriate tonicity.

In situ analysis of genetic material or a surrogate thereof: The term“in situ analysis,” as used herein means that processes provided hereinallow DNA or RNA analysis to be carried out in the same tube or on analiquot of the stopped mixture without centrifugation or extraction.That is, RNA or DNA need not be isolated from the stopped mixture priorto mixing at least a portion of the stopped mixture with a compositioncomprising reverse transcriptase or another relevant enzyme. The term“or a surrogate thereof,” as used herein means a detectable product thatrepresents the RNA or DNA present in the sample, such as an amplifiedproduct of the RNA or DNA.

Lysis Mixture: A “lysis mixture,” as used herein, comprises componentsfor isothermally lysing a sample and lacks components that can interferewith later detection of DNA or RNA, or a surrogate thereof, usingemission detection at wavelengths of 300 nm to 750 nm. A lysis mixturefor RNA analysis comprises a lysis solution and a polypeptide havingdeoxyribonuclease activity. A lysis mixture for DNA analysis lacks apolypeptide having deoxyribonuclease activity and may contain, in someembodiments, a polypeptide having ribonuclease activity. A lysisreaction is a lysis mixture combined with a sample. Incubation of alysis reaction can be for any range of time between 2 minutes to about30 minutes, about 2 minutes to about 20 minutes, about 3 minutes toabout 15 minutes, about 4 minutes to about 10 minutes, about 8 minutes,about 7 minutes, about 6 minutes, or about 5 minutes.

A lysis solution comprises, for certain embodiments herein, a Tris-baseor Tris-Cl buffer at a pH of about 7.5 to about 8.2 for a range oftemperatures such as 19° C. to 25° C., a polypeptide having proteaseactivity, and a surfactant that substantially lacks fluorescence between300 nm and 750 nm. The lysis solution is used at a lysis-effectiveconcentration. Further, the lysis solution is substantially free of acation chelator.

A Polypeptide having Protease Activity: In certain embodiments herein,the lysis solution comprises a polypeptide having protease activity suchas for example, proteinase K. In lieu of, or in addition to, proteinaseK, the lysis solution can comprise a serine protease such as trypsin,chymotrypsin, elastase, subtilisin, streptogrisin, thermitase,aqualysin, plasmin, cucumisin, or carboxypeptidase A, D, C, or Y; acysteine protease such as papain, calpain, or clostripain; an acidprotease such as pepsin, chymosin, or cathepsin; or a metalloproteasesuch as pronase, thermolysin, collagenase, dispase, an aminopeptidase orcarboxypeptidase A, B, E/H, M, T, or U.

A surfactant that substantially lacks fluorescence between 300 nm and750 nm when in use for in situ analysis of DNA, RNA or a surrogatethereof: In embodiments provided herein, the lysis solution comprises asurfactant at a concentration that has low or no emission at theemission wavelengths of dyes or labels commonly used for detecting RNAor DNA when in use for in situ analysis of DNA, RNA or a surrogatethereof.

A lysis-effective concentration of surfactant in a lysis mixture is aconcentration of surfactant at which a sample is considered fully lysedas determined by propidium iodide staining using 1% TRITON X-100™surfactant as a control. Lysis-effective concentrations of exemplarysurfactants range from 0.02% or 0.05% to 3% or more for TRITON X-114™surfactant, from 0.01% or 0.05% to 5% or more for THESIT™ surfactant,from 0.1% to 5% or more for NONIDET P40™ surfactant, and from 0.05% to1% or to 3% for TRITON X-100™ surfactant. When a combination ofsurfactants is used, the concentration of each surfactant may be loweredfrom the cited amounts.

For the methods and processes described herein, the lysate is dilutedwhen stop solution is added. The stopped mixture is further diluted whena portion is transferred to a RT-qPCR reaction. The concentration ofsurfactant in the qPCR reaction is thereby diluted when compared to theconcentration of the surfactant in the lysate. The dilution factor mayrange from a 1.25-fold dilution to a thousand-fold or more dilution.Concentrations of the above-listed surfactants that, in addition tobeing lysis-effective, have low or no emission at the emissionwavelengths of green emitters (500 nm to 549 nm) when in use for in situanalysis of RNA or a surrogate thereof include TRITON X-114™ surfactantat 0.05% to 1%; THESIT™ surfactant at 0.05% to 0.3%; TRITON X-100™surfactant at 0.05% to 0.3%; NONIDET P-40™ surfactant at 0.1% to 0.3%,or a combination thereof. Commonly used labeling dyes having emissionwavelengths of green emitters include FAM™ dye, FITC, and JOE™ dye.

A polypeptide having deoxyribonuclease activity: A polypeptide havingdeoxyribonuclease activity is present in certain lysis mixtures as setforth in embodiments herein where RNA is to be detected. The polypeptidehaving deoxyribonuclease activity is dependent upon cations such as Ca⁺⁺or Mg⁺⁺ for stability and activity. In the case where a polypeptidehaving deoxyribonuclease activity is obtained with a cation alreadypresent, which is commonly the case, additional cations are not neededin the lysis mixture. In the case where a polypeptide havingdeoxyribonuclease activity is obtained lacking cations, exogenouscations are added to the lysis mixture. A polypeptide havingdeoxyribonuclease activity can be DNase I or Nuclease BAL-31, both ofwhich are Ca⁺⁺- and Mg⁺⁺-dependent; or exonuclease I, exonuclease III,Lambda exonuclease, CviKI-1 endonuclease, or McrBC endonuclease, all ofwhich are Mg′-dependent, or an enzymatically active mutant or variantthereof. A polypeptide having deoxyribonuclease activity can be presentin the lysis mixture from 100 U/ml to 600 U/ml in some embodiments and,for other embodiments, about 200 U/ml, about 300 U/ml, about 400 U/ml,about 500 U/ml or any range of concentrations therebetween.

Substantially free of a cation chelator: In general, the lysis mixturesfor RNA sample preparation processes are substantially free of a cationchelator. A common cation chelator, such as EDTA, has been found hereinto interfere with deoxyribonuclease activity at a concentration of 1 mM.Therefore, lysis mixtures provided herein for RNA sample preparation aresubstantially free of a cation chelator, have less than about 0.1 mMcation chelator, have less than about 0.2 mM cation chelator, have lessthan about 0.5 mM or have less than 1 mM cation chelator.

Optional Lysis Mixture ingredients: In some embodiments, a calcium saltis present in the lysis mixture in concentrations ranging from 0 mM to2.5 mM for stabilizing a deoxyribonuclease. The calcium salt can be anycalcium salt that provides such function and can be calcium chloride,calcium bromide, calcium acetate, calcium formate, calcium sulfate, orcalcium phosphate, for example. In certain embodiments, the calcium saltis CaCl₂ and the CaCl₂ is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1.0mM, or 2.0 mM or any range of concentrations therebetween. In someembodiments, MgCl₂ is present in the lysis solution in concentrationsranging from 0 mM to 2.5 mM. In certain embodiments, the MgCl₂ ispresent at about 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any rangeof concentrations therebetween. Certain assays such as short tandemrepeat detection assays use lower concentrations of MgCl₂ such as about0.5 mM.

In some embodiments, the lysis mixture comprises at least one reducingagent. Use of reducing agents is well known by those of ordinary skillin the art. Exemplary reducing agents include dithiothreitol,β-mercaptoethanol, dithioerythritol, or combinations thereof.

In some embodiments, addition of a reducing agent at a finalconcentration of about 0.01 mM in the lysis mixture together withaddition to the stop solution (as discussed below) improves cyclethreshold values.

In some embodiments, the lysis mixture further comprises at least oneadditional catabolic enzyme. For example, a glycoside hydrolase such asamylase, lysozyme or cellulase can be included for degradation ofpolysaccharides, or lipase may be included for degradation of lipids, ora combination thereof may be used. In such cases, it may be necessary tobalance the concentration, reaction conditions, or timing of addition ofone or more catabolic enzymes, in order to prevent degradation of the atleast one additional catabolic enzyme by the protease. Such reactionoptimization is well within the skill of those of ordinary skill in theart in light of the teachings herein.

Exemplary non-limiting embodiments of lysis mixtures are prepared byobtaining stock solutions of 1M Tris-base pH 8.0, 1M MgCl₂, 1M CaCl₂, 1MDTT, proteinase K at 20 mg/ml, 20% TRITON X-114™ and nuclease-freewater. Stock solutions are diluted to form a lysis solution of Tris pH8.0, 10 mM; MgCl₂, 0.5 mM; CaCl₂, 0.5 mM; a reducing agent such as DTT,β-mercaptoethanol or dithioerythritol, 0.01 mM; protease such asproteinase K, 100 ug/ml; and TRITON X-114™, 0.1%, in nuclease freewater. The pH is adjusted to pH 7.8+/−0.1 with HCl at a temperature of19° C.-25° C. (a range of pH values is about pH 7.5 to about pH 8.2).The lysis solution can be stored at −20° C., at 4° C., and has beenfound to be stable at 25° C. for one year. In some embodiments, lysiscan be carried out in a 50 uL volume at a pH of about 7.8.

Stop Mixture: In some embodiments, a stop mixture comprises a cationchelator effective to inactivate the polypeptide havingdeoxyribonuclease activity of the lysis mixture, an inhibitor of thepolypeptide having protease activity of the lysis mixture, andgenerally, a stop mixture comprises a Tris-base or Tris-Cl buffer atabout pH 8. For analysis embodiments by RT-PCR, the stopped mixture iscompatible with reverse transcriptase and DNA polymerase reactionconditions. A stopped mixture can be included in such reactions up to45% or up to 65% or more of the RT or PCR reaction volume depending uponthe concentrations of the various components.

A cation chelator effective to inactivate the polypeptide havingdeoxyribonuclease activity of the stop mixture: For embodiments wherethe polypeptide having deoxyribonuclease activity is dependent uponcalcium ions for stability and activity, the cation chelator comprises acalcium chelator such as EGTA, EDTA, or citrate, for example. Forembodiments where the polypeptide having deoxyribonuclease activity isdependent upon magnesium ions for stability and activity, the cationchelator comprises a magnesium chelator such as EDTA, for example. Ofcourse, divalent cation chelators bind a variety of divalent cations andoverlap in specificity for divalent cations is expected. Cationchelators include EGTA, ethylenediamine tetraacetic acid (EDTA), sodiumcitrate, cation exchange beads such as SP SEPHAROSE™ beads (GEHealthcare), 1,10-phenanthroline,tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or acombination thereof. EGTA inhibits DNase I at 4 mM and is compatiblewith RT-PCR.

An inhibitor of the polypeptide having protease activity: The stopmixture comprises a chemical or molecular inhibitor of the polypeptidehaving protease activity. In addition, the inhibitor has essentially noinhibitory effect on reverse transcriptase or on DNA polymerase.

For some embodiments where the polypeptide having protease activity isproteinase K, the chemical inhibitor comprises amethoxysuccinyl-Ala-Ala-Pro/Leu-Ala/Val-haloalkyl ketone (SEQ ID NO:2)where the halo is chloro, fluoro, iodo or bromo and the alkyl is C₁ toC₃ or active derivatives or analogs thereof. In certain embodiments, thechemical inhibitor comprisesmethoxysuccinyl-Ala-Ala-Pro/Leu-AlaNal-chloroalkyl ketone (SEQ ID NO:2)where the alkyl is C₁ to C₃ or active derivatives or analogs thereof.Methoxysuccinyl-AAPV-chloromethyl ketone (SEQ ID NO:1) (Bachem,Torrance, Calif., or, for example, in DMSO, Sigma-Aldrich, St. Louis,Mo.) at a concentration as low as 0.75 mM was demonstrated to haveinhibitory activity for 100 μg/ml PK and is compatible with bothone-step and two-step RT-PCR reactions.

Other inhibitors of proteinase K include carbobenzoxy-Ala-Ala-COCH₂Cl,carbobenzoxy-Ala-Ala-Phe-COCH₂Cl or carbobenzoxy-Phe-Pro-Arg-COCH₂Cl asdescribed by Wolf et al. (JBC 266:26, 17695, 1991), andphenylmethylsulfonyl fluoride (PMSF). In some embodiments, PMSF ispresent at a concentration of up to 2 mM in the stopped mixture or up to400 uM in an RT reaction. Further inhibitors of the polypeptide havingprotease activity include the proline-containing tetrapeptidederivatives and proline-containing tripeptide derivatives of U.S. Pat.No. 4,596,789 (application Ser. No. 603,408 filed Apr. 24, 1984) andU.S. Pat. No. 4,691,007 (application Ser. No. 806,265 filed Dec. 6,1985) issued Jun. 24, 1986 and Sep. 1, 1987, respectively, to Anand S.Dutta et al., including acid- and base-addition salts thereof; thepeptide derivatives of U.S. Pat. No. 4,910,190 (application Ser. No.5,538 filed Jan. 20, 1987) and issued Mar. 20, 1990 to Bergeson et al.including salts thereof; the peptide derivatives of U.S. Pat. No.5,414,132 (application Ser. No. 940,932 filed Sep. 4, 1992) issued May9, 1995, and U.S. Pat. No. 5,907,068 (application Ser. No. 07/941,001filed Sep. 4, 1992) issued May 25, 1999, both to Stein et al. includingsalts thereof; and the peptide derivatives of U.S. Pat. No. 5,055,450(application Ser. No. 493,025 filed Mar. 13, 1990) issued Oct. 8, 1991,and U.S. Pat. No. 5,726,158 (application Ser. No. 467,333 filed Jun. 6,1995) issued Mar. 10, 1998, both to Edwards et al. including saltsthereof.

Further protease/protease inhibitor pairs include leupeptin as aninhibitor for serine and cysteine proteases such as plasmin, trypsin,papain, kallikrein and cathepsin B; 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF) as an inhibitor for serine proteases such aschymotrypsin, kallikrein, plasmin, thrombin, and trypsin; aprotinin asan inhibitor of serine proteases such as trypsin, chymotrypsin, plasminand kallikrein; benzamidine as an inhibitor of trypsin; N-acetyl eglin-Cas an inhibitor of chymotrypsin, subtilisin, leukocyte elastase andcathepsin G; and antipain or plasmin for inhibition of a serine orcysteine such as papain and trypsin.

Further protease inhibitors include aptamers, or polyclonal ormonoclonal antibodies having binding affinity and binding specificityfor the polypeptide having protease activity of the lysis mixture.

Optional Stop Mixture Ingredients: In some embodiments, the stop mixturecomprise one or more ribonuclease inhibitors such as placentalribonuclease inhibitor protein (RIP, Promega, Madison, Wis.) at about0.2 U/uL to about 0.002 U/ul, SUPERase-In™ (protein-based inhibitor forRNase A, B, C, 1, and T1, Catalog No. AM2694, AMBION®, Austin, Tex.),RNase inhibitor (a recombinant human placental protein having inhibitoryactivity for neutral pancreatic RNase A-type enzymes, Catalog No.AM2682, AMBION®, Austin, Tex.) and anti-RNase A (protein-based inhibitorfor RNase A, Catalog No. AM2690, AMBION®, Austin, Tex.). The addition ofRIP reduces PCR cycle threshold values at 30 min for both 5000 and100,000 cell samples. Final RIP concentration (0.2 U/ul, after additionto lysate; 2.2 U/uL in the stop solution) helps prevent RNA degradation,particularly if the lysate is allowed to sit at room temperature forlonger than about 20 minutes.

As recited supra, addition of a reducing agent to the stop solution at0.11 mM improved cycle threshold results for PCR at 10 minutespost-stop. While not wanting to be bound by theory, a reducing agent isprovided for the stop solution to improve functionality and stability ofribonuclease inhibitor protein (RIP).

A stop reaction is incubated for up to 2 minutes. After about 20minutes, PCR cycle threshold values increase very gradually. In someembodiments, a stopped mixture has a pH of 7.3-7.8 as a result of theprotease inhibition reaction.

Detection of DNA, RNA or a surrogate thereof: Embodiments of detectingDNA, RNA or a surrogate thereof in a stopped mixture as provided hereinincludes detection means using emission by an emitter that isrepresentative of the RNA or DNA in the stopped mixture.

In some embodiments, RNA of a stopped mixture as provided by teachingsherein is detected in situ by adding or mixing at least a portion of thestopped mixture with a composition comprising reverse transcriptase toform a reverse transcriptase reaction mixture. A reverse transcriptionreaction provides a surrogate of the RNA that can be detectable. Anyreverse transcriptase known to those of ordinary skill in the art can beused such as, for example, MMLV-RT (murine maloney leukemiavirus-reverse transcriptase), avian myelogenous virus reversetranscriptase (AMV-RT), human immunodeficiency virus (HIV)-RT and theTth DNA polymerase which has reverse transcriptase activity if Mn⁺⁺ isprovided.

A positive control for detection of RNA or DNA can be a non-homologousRNA random sequence such as XENOTMRNA (Applied Biosystems, Foster City,Calif.). A control for qPCR can be a β-actin probe/primer set, alsoavailable from Applied Biosystems, for example. The positive control canbe mixed with the stop solution and therefore is added at the time ofadding stop solution to a sample.

Amplification: As used herein, “amplification” or “amplify” and the likerefers to a process that results in an increase in the copy number of amolecule or set of related molecules. As the term applies to a stoppedmixture herein, amplification means the production of multiple copies ofthe target nucleic acid, a surrogate of a target nucleic acid, or aportion thereof. Amplification can encompass a variety of chemical andenzymatic processes such as a polymerase chain reaction (PCR), a stranddisplacement amplification reaction, a transcription mediatedamplification reaction, or a nucleic acid sequence-based amplificationreaction, for example. Following at least one amplification cycle, theamplification products can be detected or can be separated from at leastone other component of the amplification mixture based on theirmolecular weight or length or mobility prior to detection.

Polymerase Chain Reaction: PCR includes introducing a molar excess oftwo or more extendable oligonucleotide primers to a reaction mixturecomprising the stopped mixture where the primers hybridize to oppositestrands of a DNA, RNA or RNA surrogate. The reaction mixture issubjected to a program of thermal cycling in the presence of a DNApolymerase, resulting in the amplification of the DNA or RNA surrogatesequence flanked by the primers. Reverse transcriptase PCR is a PCRreaction that uses an RNA template and a reverse transcriptase, or apolypeptide having reverse transcriptase activity, to first generate asingle stranded DNA molecule prior to the multiple cycles ofDNA-dependent DNA polymerase primer elongation as cited above. Methodsfor a wide variety of PCR applications are widely known in the art, anddescribed in many sources, for example, Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., NewYork (1994).

Criteria for designing sequence-specific primers are well known topersons of ordinary skill in the art. Detailed descriptions of primerdesign that provide for sequence-specific annealing can be found, amongother places, in Diffenbach and Dveksler, PCR Primer, A LaboratoryManual, Cold Spring Harbor Press, 1995, and Kwok et al. (Nucl. Acid Res.18:999-1005, 1990). The sequence-specific portions of the primers are ofsufficient length to permit specific annealing to complementarysequences, as appropriate. A primer does not need to have 100%complementarity with a primer-specific portion for primer extension tooccur. Further, a primer can be detectably labeled such that the labelis detected by spectroscopy. A primer pair is sometimes said to consistof a “forward primer” and a “reverse primer,” indicating that they areinitiating nucleic acid polymerization in opposing directions fromdifferent strands of a duplex template.

In some embodiments, a primer as set forth herein can comprise auniversal priming sequence. The term “universal primer” refers to aprimer comprising a universal sequence that is able to hybridize to all,or essentially all, potential target sequences in a multiplexedreaction. The term “semi-universal primer” refers to a primer that iscapable of hybridizing with more than one (e.g., a subset), but not all,of the potential target sequences in a multiplexed reaction. The terms“universal sequence,” “universal priming sequence” or “universal primersequence” or the like refer to a sequence contained in a plurality ofprimers, where the universal priming sequence that is found in a targetis complementary to a universal primer.

For real time PCR, a passive reference dye, ROX™ dye, can be included inPCR reactions to provide an internal reference to which the reporter-dyesignal can be normalized during data analysis. Normalization can beaccomplished using Applied Biosystems' Sequence Detection System (SDS)software.

In certain embodiments, single-stranded amplification products can begenerated by methods including, without limitation, asymmetric PCR,asymmetric reamplification, nuclease digestion, and chemicaldenaturation. For example, single-stranded sequences can be generated bycombining at least one first primer or at least one second primer from aprimer set, but not both, in an amplification reaction mixture, or bytranscription, for example, when a promoter-primer is used in a firstamplification mixture, a second amplification mixture, or both.

Polymerase: The term “polymerase,” as used herein, refers to apolypeptide that is able to catalyze the addition of nucleotides oranalogs thereof to a nucleic acid in a template dependent manner, forexample, the addition of deoxyribonucleotides to the 3′-end of a primerthat is annealed to a nucleic acid template during a primer extensionreaction. Nucleic acid polymerases can be thermostable or thermallydegradable. Suitable thermostable polymerases include, but are notlimited to, polymerases isolated from Thermus aquaticus, Thermusthermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcuslitoralis, and Thermotoga maritima. Suitable thermodegradablepolymersases include, but are not limited to, E. coli DNA polymerase I,the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5DNA polymerase, T7 DNA polymerase, and others. Examples of otherpolymerizing enzymes that can be used in the methods described hereininclude but are not limited to T7, T3, SP6 RNA polymerases; and AMV,M-MLV and HIV reverse transcriptases.

Commercially available polymerases include, but are not limited toAMBION'S SUPERTAQ®, TAQFS®, AMPLITAQ® CS (Applied Biosystems), AMPLITAQ®FS (Applied Biosystems), KENTAQ1® (AB Peptide, St. Louis, Mo.),TAQUENASE® (Scien Tech Corp., St. Louis, Mo.), THERMOSEQUENASE®(Amersham), Bst polymerase, READERTMTaq DNA polymerase, VENT® DNApolymerase, VENT_(R)® DNA Polymerase, VENT_(R)® (exo⁻) polymerase andDEEPVENT® DNA polymerase, (all VENT® polymerases can be obtained fromNew England Biolabs), PFUTurbo™ DNA polymerase (Stratagene), Pwopolymerase, Tth DNA polymerase, KlenTaq-1 polymerase, SEQUENASE™ 1.0 DNApolymerase (Amersham Biosciences), SEQUENASE™ 2.0 DNA polymerase (UnitedStates Biochemicals), and an enzymatically active mutant and variantthereof.

Descriptions of DNA polymerases can be found in, among other places,Lehninger Principles of Biochemistry, 3d ed., Nelson and Cox, WorthPublishing, New York, N.Y., 2000, particularly Chapters 26 and 29;Twyman, Advanced Molecular Biology: A Concise Reference, Bios ScientificPublishers, New York, N.Y., 1999; Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., including supplementsthrough May 2005 (hereinafter “Ausubel et al.”); Lin and Jaysena, J.Mol. Biol. 271:100-11, 1997; Pavlov et al., Trends in Biotechnol.22:253-60, 2004; and Enzymatic Resource Guide: Polymerases, 1998,Promega, Madison, Wis.

In various detection embodiments, amplification is optionally followedby additional steps, for example, but not limited to, labeling,sequencing, purification, isolation, hybridization, size resolution,expression, detecting and/or cloning. In certain embodiments, one orboth PCR primers can comprise a label, such as, for example, afluorophore. A label can facilitate detection of an amplificationproduct comprising a labeled PCR primer. In various detectionembodiments, following the PCR, biotinylated strands can be captured,separated, and detected.

Multiplex Assays: The term “multiplex assays” refers to PCR reactionsthat use more than two primers in a single reaction and at the same timeso that more than one different amplified product is produced anddetected. For example, more than two pair of amplification primers arecontacted at the same time and/or in the same solution. Several targetRNAs or DNAs can be detected simultaneously using multiplex assays. Amultiplex reaction can also include a multiplicity of singleplex PCRreactions run in parallel, e.g., the TAQMAN® Low Density Array (TLDA).Sample preparation processes described herein have been demonstrated tobe compatible with multiplex assays.

Real-time PCR: As used herein, “real-time PCR” refers to the detectionand quantitation of a DNA, a RNA or a surrogate thereof in a sample. Insome embodiments, the amplified segment or “amplicon” can be detectedusing a 5′-nuclease assay, particularly the TAQMAN® assay as describedby e.g., Holland et al. (Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991);and Heid et al. (Genome Research 6:986-994, 1996). For use herein, aTAQMAN® nucleotide sequence to which a TAQMAN® probe binds can bedesigned into the primer portion, or known to be present in a RNA or aDNA of a sample.

“T_(m)” refers to the melting temperature (temperature at which 50% ofthe oligonucleotide is a duplex) of an oligonucleotide determinedexperimentally or calculated using the nearest-neighbor thermodynamicvalues of Breslauer et al. (Proc. Natl. Acad. Sci. USA 83:3746 3750,1986) for DNA or Freier et al. (Proc. Natl. Acad. Sci. USA 83:9373-9377,1986) for RNA. In general, the T_(m) of the TAQMAN® probe is about 10degrees above the T_(m) of amplification primer pairs. Amplificationprimer sequences and double dye-labeled TAQMAN® probe sequences can bedesigned using PRIMER EXPRESS™ (Version 1.0, Applied Biosystems, FosterCity, Calif.) or mFOLD™ software (now UNIFold™) (IDT, San Jose, Calif.).

When a TAQMAN® probe is hybridized to DNA, RNA or a surrogate thereof,the 5′-exonuclease activity of a thermostable DNA-dependent DNApolymerase such as SUPERTAQ® (a Taq polymerase from Thermus aquaticus,Ambion, Austin, Tex.) digests the hybridized TAQMAN® probe during theelongation cycle, separating the fluor from the quencher. The reporterfluor dye is then free from the quenching effect of the quencher moietyresulting in a decrease in FRET and an increase in emission offluorescence from the fluorescent reporter dye. One molecule of reporterdye is generated for each new molecule synthesized, and detection of thefree reporter dye provides the basis for quantitative interpretation ofthe data. In real-time PCR, the amount of fluorescent signal ismonitored with each cycle of PCR. Once the signal reaches a detectablelevel, it has reached the “threshold or cycle threshold (Ct).” Afluorogenic PCR signal of a sample can be considered to be abovebackground if its Ct value is at least 1 cycle less than that of ano-template control sample. The term “Ct” represents the PCR cyclenumber when the signal is first recorded as statistically significant.Thus, the lower the Ct value, the greater the concentration of nucleicacid target. In the TAQMAN® assay, typically each cycle almost doublesthe amount of PCR product and therefore, the fluorescent signal shoulddouble if there is no inhibition of the reaction and the reaction wasnearly 100% efficient with purified nucleic acid. Certain systems suchas the ABI 7700 and 7900HT Sequence Detection Systems (AppliedBiosystems, Foster City, Calif.) conduct monitoring during each thermalcycle at a pre-determined or user-defined point.

Detection method embodiments using a TAQMAN® probe sequence comprisecombining the stopped mixture or the reverse transcribed mixture withPCR reagents, including a primer set having a forward primer and areverse primer, a DNA polymerase, and a fluorescent detectoroligonucleotide TAQMAN® probe, as well as dNTP's and a salt, to form anamplification reaction mixture; subjecting the amplification reactionmixture to successive cycles of amplification to generate a fluorescentsignal from the detector probe; and quantitating the nucleic acidpresence based on the fluorescent signal cycle threshold of theamplification reaction.

Protocols and reagents for means of carrying out further 5′-nucleaseassays are well known to one of skill in the art, and are described invarious sources. For example, 5′-nuclease reactions and probes aredescribed in U.S. Pat. No. 6,214,979 issued Apr. 10, 2001; U.S. Pat. No.5,804,375 issued Sep. 8, 1998; U.S. Pat. No. 5,487,972 issued Jan. 30,1996; and U.S. Pat. No. 5,210,015 issued May 11, 1993, all to Gelfand etal.

In various embodiments, a detection method can utilize any probe thatcan detect a nucleic acid sequence. In some configurations, a detectionprobe can be, for example, a TAQMAN® probe described supra, a stem-loopmolecular beacon, a stemless or linear beacon, a PNA MOLECULAR BEACON™,a linear PNA beacon, non-FRET probes, SUNRISE®/AMPLIFLUOR® probes,stem-loop and duplex SCORPION™ probes, bulge loop probes, pseudo knotprobes, cyclicons, MGB ECLIPSE™ probe, a probe complementary to aZIPCODE™ sequence, hairpin probes, peptide nucleic acid (PNA) light-upprobes, self-assembled nanoparticle probes, and ferrocene-modifiedprobes as known by one of ordinary skill in the art. A detection probehaving a sequence complementary to a detection probe hybridizationsequence, such as a ZIPCODE™ sequence, a fluorphore and a mobilitymodifier can be, for example, a ZIPCHUTE™ probe supplied commercially byApplied Biosystems (Foster City, Calif.).

Label or Reporter: A “label” or “reporter,” as used herein, refers to amoiety or property that allows the detection of that with which it isassociated and, for use herein, has emission spectra at between andincluding 300 nm to 750 nm. In certain embodiments, the emission spectrais at less than about 499 nm such as for blue emitters such as certainAlexa Fluor emitters, Cascade Blue, and Pacific Blue; at 500 nm to 549nm emitters such as for green emitters such as certain Alexa Fluoremitters, BODIPY FL, fluorescein (FITC), CYANINE™ 2 dye, Catskill Green,5-FAM™ dye, 6-FAM™ dye, succinimidyl ester, JOE™ dye, MFP488, the OregonGreen emitters and TET™ dye; at 550 nm to 584 nm emitters such as yellowemitters such as certain Alexa Fluor emitters, CYANINE™ 3 dye, HEX™ dye,NED™ dye, R-Phycoerythrin (R-PE), 5-TAMRA™ dye, TRITC (Rhodamine), andVIC® dye; at 585 nm to 615 nm emitters such as orange emitters such ascertain Alexa Fluor emitters, CYANINE™ 3.5 dye, Lissamine Rhodamine,ROX™ dye, and R-Phycoerythrin-TEXAS RED® dye; and at 616 nm to 700 nmemitters such as red emitters such as certain Alexa Fluor emitters,CYANINE™ 5 dye, Quantum Red, Rodamine Red-X, and TEXAS RED® dye.

The label can be attached covalently or non-covalently to a DNA product,to a RNA product, or to a surrogate thereof such as an amplicon thereof.Commonly used labels include dyes that are negatively charged, such asdyes of the fluorescein family including, e.g. FAM™ dye, HEX™ dye, TET™dye, JOE™ dye, NAN and ZOE; or dyes that are neutral in charge, such asdyes of the rhodamine family including, e.g., TEXAS RED® dye, ROX™ dye,R110, R6G, and TAMRA™ dye; or dyes that are positively charged, such asdyes of the CYANINE™ family including e.g., Cy™2 dye, Cy™3 dye, Cy™5dye, Cy™5.5 dye and Cy™7 dye. FAM™ dye, HEX™ dye, TET™ dye, JOE™ dye,NAN, ZOE, ROX™ dye, R110, R6G, and TAMRA™ dyes are available from, e.g.,Applied Biosystems (Foster City, Calif.) or Perkin-Elmer, Inc.(Wellesley, Mass.); TEXAS RED® dye is available from, e.g., MolecularProbes, Inc. (Eugene, Oreg.); and Cy™2 dye, Cy™3 dye, Cy™5 dye, Cy™5.5dye and Cy™7 dye, and are available from, e.g., Amersham BiosciencesCorp. (Piscataway, N.J.). In certain amplification embodiments, thefluorescer molecule is a fluorescein dye and the quencher molecule is arhodamine dye.

A label or reporter can comprise both a fluorophore and a fluorescencequencher. The fluorescence quencher can be a fluorescent fluorescencequencher, such as the fluorophore TAMRA™ dye, or a non-fluorescentfluorescence quencher (NFQ), for example, a combined NFQ-minor groovebinder (MGB) such as an MGB ECLIPSE™ minor groove binder supplied byEpoch Biosciences (Bothell, Wash.) and used with TAQMAN™ probes (AppliedBiosystems, Foster City, Calif.). The fluorophore can be any fluorophorethat can be attached to a nucleic acid, such as, for example, FAM™ dye,HEX™ dye, TET™ dye, JOE™ dye, NAN, ZOE, TEXAS RED® dye, ROX™ dye, R110,R6G, TAMRA™ dye, Cy™2 dye, Cy™3 dye, Cy™5 dye, Cy™5.5 dye and Cy™7 dyeas cited above as well as VIC® dye, NED™ dye, LIZ® dye, ALEXA, Cy™9 dye,and dR6G.

Further examples of labels include black hole quenchers (BHQ)(Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), andDabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).

Labels can also comprise sulfonate derivatives of fluorescein dyes,phosphoramidite forms of fluorescein, phosphoramidite forms of CY™5 dye(available for example from Amersham), and intercalating labels such asethidium bromide, SYBR™ Green I dye and PICOGREEN™ dye (MolecularProbes). Generally, an intercalating label is a molecule that reversiblyinserts between two other molecules (or groups) such as between thebases of DNA.

In various embodiments, qPCR reactions can include master mixes such asthe TAQMAN® Gene Expression Master Mix, TAQMAN® Universal PCR MasterMix, TAQMAN® Fast Universal PCR Master Mix, Power SYBR® Green PCR MasterMix, Fast SYBR® Green Master Mix, TAQMAN® RNA-to-C_(T)™ 1-Step Kit, andthe Power SYBR® Green RNA-to-C_(T)™ 1-Step Kit, for example, all fromApplied Biosystems.

In various embodiments, detection of emission such as fluorescence canbe by any method known to skilled artisans, and can include, forexample, real time detection for PCR or end point detection. Detectionof fluorescence, for example, can be qualitative or quantitative.Quantitative results can be obtained, for example, with the aid of afluorimeter, for example a fluorimeter as part of an integrated nucleicacid analysis system, such as, for example, an Applied Biosystems ABIPRISM™ 7900HT Sequence Detection System. Furthermore, quantitativeresults can be obtained in some configurations using a real-time PCRanalysis. Some non-limiting examples of protocols for conductingfluorogenic assays such as TAQMAN® assays, including analytical methodsfor performing quantitative assays, can be found in publications suchas, for example, “SNPLEX™ Genotyping System 48-plex”, AppliedBiosystems, 2004; “User Bulletin #2 ABI PRISM™ 7700 Sequence DetectionSystem,” Applied Biosystems 2001; “User Bulletin #5 ABI PRISM™ 7700Sequence Detection System,” Applied Biosystems, 2001; and “Essentials ofReal Time PCR,” Applied Biosystems (Foster City, Calif.). FluorogenicPCR assays used in some configurations of the present teachings can beperformed using an automated system, such as, for example, an ABI 7700Sequence Detection System (Applied Biosystems).

In some embodiments, detection can be achieved using microarrays or beadarrays and related software, such as the Applied Biosystems Array Systemwith the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer,and other commercially available array systems available fromAffymetrix, Agilent, and Illumina, among others (see also Gerry et al.,J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, includingsupplements, 2003).

Further method embodiments for detection of DNA, RNA, or a surrogatethereof comprise use of a promoter sequence or a complement thereof andthe method includes combining the DNA, RNA, or a surrogate thereof withPCR reagents, including at least one primer set and a DNA polymerase, toform a first amplification reaction mixture subjecting the firstamplification reaction mixture to at least one cycle of amplification togenerate a first amplification product comprising the promoter sequence;combining the first amplification product with an RNA polymerase and aribonucleoside triphosphate solution comprising at least one of rATP,rCTP, rGTP, rUTP, or aminoallyl-rUTP to form a transcription reactionmixture; incubating the transcription reaction mixture under appropriateconditions to generate an RNA transcription product; and detectingpresence of the target nucleic acid by detection of the RNAtranscription product or a portion thereof. In certain embodiments, thepolymerase is reverse transcriptase.

Exemplary RNA polymerases include T7, T3, or SP6 RNA polymerase andexemplary promoters include the T7, T3, or SP6 promoters. The RNAtranscription product or a portion thereof can be detected using, forexample, the aminoallyl-rUTP which is available for coupling to asuccinimide ester label for detection.

Enzymatically Active Mutants or Variants Thereof: The term“enzymatically active mutants or variants thereof” when used inreference herein to an enzyme such as a protease, deoxyribonuclease, apolymerase or the like, refers to a polypeptide derived from thecorresponding enzyme that retains at least some of the desired enzymaticactivity. Enzymatically active mutants or variants include, for example,fragments, recombinantly expressed fragments, naturally-occurringmutants, mutants generated using mutagens, genetically engineeredmutants, mutants due to amino acid insertions or deletions or due tonucleic acid nonsense, missense, or frameshift mutations, reversiblymodified enzymes, splice variants, polypeptides having modificationssuch as altered glycosylation, disulfide bonds, hydroxyl side chains,and phosphate side chains, or crosslinking, and the like. Protocols formeasuring enzymatic activity using an appropriate assay are known to oneof ordinary skill in the art.

Cell lysates provided herein are useful for any method of detection ofnucleic acid that uses a dye that has a detectable emission. Inparticular, a dye or label that fluoresces in the 500 nm to 615 nm rangesuch as used in PCR, RT-PCR, qRT-PCR, siRNA-mediated gene knockdown,high-throughput assessment of any kind particularly in 96-well or384-well plates is envisioned for use herein. Samples can be processeddirectly in culture plates, minimizing sample handling and the potentialfor sample loss or transfer error. The cell lysis protocol in 384-wellplates is readily automated on robotic platforms. cDNA can then besynthesized directly from the lysate using the High Capacity cDNA RTKit, or the High Capacity RNA-to-cDNA kit, and real-time PCR performedusing the TAQMAN® Gene Expression Master Mix (Applied Biosystems, FosterCity, Calif.) on the 7900HT Real Time PCR System. Custom libraries ofSilencer® Pre-designed siRNAs and TAQMAN® Gene Expression Assays platedto specification in 384-well plates can be obtained directly from themanufacturer (Applied Biosystems). Processes provided by the teachingsherein ensure high-throughput processing, efficient use of reagents andinstruments, a minimal amount of hands-on time, and accurate andreliable results.

Kits: A “kit,” as used herein, refers to a combination of items forperforming a sample preparation process as set forth herein. Embodimentsof kits comprise, for example, lysis mixture components and stop mixturecomponents. Lysis mixture components comprise a polypeptide havingprotease activity, a surfactant comprising TRITON X-114™, THESIT™,TRITON X-100™, NONIDET P-40™, or a combination thereof, and apolypeptide having deoxyribonuclease activity. The lysis mixturecomponents are substantially free of a cation chelator. Stop mixturecomponents comprise a cation chelator, and an inhibitor of thepolypeptide having protease activity. Components of kits may be packagedtogether or separately as desired for the processes described herein.

Kit embodiments can further comprise reagents for reverse transcription,such as reverse transcriptase, a reverse primer, dNTPs or a reversetranscriptase buffer, or can further comprise reagents for PCR, such asa DNA polymerase, for example.

Embodiments of kits can further comprise a detector probe such as a5′-nuclease probe such as a TAQMAN® probe, an RNA or a DNA controlnucleic acid, reagents for sample collection, an RNA polymerase or anenzymatically active mutant or variant thereof, or ribonucleotides rATP,rCTP, rGTP, rUTP, or aminoallyl-rUTP. In certain kit embodiments,amplification primers can be attached to a solid support such as amicroarray.

In some kit embodiments, an enzyme comprising reverse transcriptaseactivity and thermostable DNA-dependent DNA polymerase activity are thesame enzyme, e.g., Thermus sp. ZO5 polymerase or Thermus thermophiluspolymerase.

When components of a kit are provided in one and/or more liquidsolutions, the liquid solution comprises an aqueous solution that can bea sterile aqueous solution. In some embodiments, at least one componentof the kit can be provided as a dried powder. When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventcan also be provided in another container means. The container meanswill generally include at least one vial, test tube, flask, bottle,syringe and/or other container means, into which the solutions areplaced, and in some embodiments, suitably aliquoted. The kits can alsocomprise a further container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

A kit can also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionscan include variations that can be implemented.

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLE 1 Effect of EDTA on Preparation of Samples for Nucleic AcidAnalysis

Studies were conducted on the effect of EDTA on DNase I and proteinase Kactivity for preparation of samples for nucleic acid analysis. DNase I(0.2 U) was mixed with 10 uL DNaseALERT™ solution (catalog no. AM1970,AMBION® Inc., Austin Tex.) in Buffer A (10 mM Tris pH 7.5, 3 mM MgCl₂, 1mM CaCl₂) with 1 mM EDTA (FIG. 2, ♦, diamonds), Buffer A without EDTA(FIG. 2, ●, circles), or DNase I buffer (FIG. 2, ▪, squares, 10 mM TrispH 7.5, 2.5 mM MgCl₂, 0.5 mM CaCl₂) in a final volume of 100 ul. The 590nm fluorescence intensity with 544 nm excitation was measured with time.

FIG. 2 provides data demonstrating that 1 mM EDTA appears to have aninhibitory effect on DNase I activity using the DNaseALERT™ systemassay. In addition, the DNase I buffer having less Mg⁺⁺ and less Ca⁺⁺provided slightly better results than Buffer A.

In a separate study, proteinase K activity was shown to be independentof EDTA concentration.

EXAMPLE 2 Effect of Surfactant on Preparation of Samples for NucleicAcid Analysis

Lysis efficacies of seven surfactants were tested by PI staining (DNAfluorochrome propidium iodide stain for viability, 1 mg/ml) of 10⁵ HeLacells treated with 0.01% to 5% surfactant. The buffer was 10 mM Tris pH7.5, 2.5 mM MgCl₂, 0.5 mM CaCl₂.

TWEEN™ 20, TWEEN™ 40, and TWEEN™ 80 were not effective at lysing cellsat concentrations up to 3%. NONIDET™ P-40 (Roche, Mannheim, Germany) waseffective for lysis at concentrations at and above 0.1% to 5%. THESIT™was effective for lysis at concentrations at and above 0.05% to 5%.TRITON™ X-114 (Dow Chemical) was effective for lysis at concentrationsat and above 0.1% to 3%. TRITON™ X-100 (Dow Chemical) was shown to beeffective for lysis at concentrations at and above 0.05%; a 1% solutionwas considered as a control for full lysis. Results using TRITON™ X-114surfactant at six different concentrations and using TRITON X-100™surfactant at one concentration as determined by propidium iodide (PI)staining (1 mg/ml) of 10⁵ HeLa cells are provided in FIG. 3.

The effects of surfactants on PCR reactions were then studied. qPCR (20uL) reactions with 4 ng Human Universal reference liver cDNA (CatalogNo. 780622; STRATAGENE®, La Jolla, Calif.) were performed by adding 2 uLof surfactant resuspended in 10 mM Tris pH 7.5 at concentrations of0.0%, 0.01%, 0.03%, 0.1%, 0.3%, and 1%. qPCR reactions were carried outusing the TAQMAN® Universal PCR Master Mix (Applied Biosystems CatalogNo. 4304437), and qRT-PCR reactions were carried out using the TAQMAN®One-Step RT-PCR Master Mix (Applied Biosystems Catalog No. 4309169).TAQMAN® gene expression assays were used to determine expressions levelsof genes PPIA (peptidylprolyl isomerase A (cyclophilin A)) and B2M(β-2-microglobulin).

THESIT™, TRITON X-100™ and NONIDET P-40™ had negative effects on PCR asdemonstrated by an increase in Ct at concentrations at or above 0.3%(0.03% final in PCR) as shown by the data of FIG. 4 (THESIT™, diamonds,dashed line; TRITON X-100™, squares, dotted line; and NONIDET P-40™,circles, solid line). TRITON X-114™ had no effect on qPCR, even at 1%(0.1% final in PCR) as shown by the data of FIG. 4 (triangles, solidline). This effect was seen with TAQMAN® Universal PCR Master Mix(Applied Biosystems Catalog No. 4304437) that uses AMPLITAQ GOLD® DNApolymerase. These results may be due to an increase in cloudiness of thesolution and/or an increase in FAM™ background fluorescence (Ro) asshown by the data of FIG. 5 (TRITON X-114™ (triangles, solid line),THESIT™ (diamonds, dashed line), TRITON X-100™ (squares, dotted line),and NONIDET P-40™ (circles, solid line)). ROX™ background fluorescencewas not affected.

Each of FIG. 6A-FIG. 6D provides an amplification plot of Rn(fluorescence corrected to a reference dye, ROX™) vs cycle number in thepresence of one of four surfactants and at concentrations cited for dataof FIG. 4 and FIG. 5. The fluor used in the PCR reaction was FAM™ andthe quencher was MGB™. THESIT™ (data of FIG. 6A) increased Ro(background fluorescence) but had little effect on Rn. TRITON X-100™(data of FIG. 6B) and NONIDET P-40™ (data of FIG. 6D) increasedbackground fluorescence and decreased Rn. TRITON X-114™ (data of FIG.6C) slightly increased background fluorescence and had little to noeffect on Rn at lower concentrations. For use with FAM™ dye detection,TRITON X-114™ appears to be an exemplary surfactant for use in the lysisbuffer.

In separate studies, the surfactants cited above were found to becompatible with DNase I and proteinase K activity.

EXAMPLE 3 Inactivation of Lysis Enzymes for Preparation of Samples forNucleic Acid Analysis

Some embodiments herein provide for chemical inhibition of proteinase Kand of DNase I so as to provide the isothermal characteristics ofcertain preparation methods herein. In some embodiments, such chemicalinhibition is also designed to be compatible with RT-PCR.

PMSF (phenylmethylsulfonylfluoride), AAPV(methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, SEQ ID NO:1), TLCK(N-tosyl-L-lysine-chloromethyl ketone), leupeptin (Ac-Leu-Leu-Arg-CHO),AEBSF, N-Acetyl-eglin C (Catalog No. E7888; Sigma-Aldrich, St. LouisMo.), aprotinin (also known as bovine pancreatic trypsin inhibitor,BPTI), benzamidine, BSA (bovine serum albumin), antipaindihydrochloride, and AAPA (methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethylketone, SEQ ID NO:3) were studied for inhibition of proteinase K (PK) atroom temperature and for compatibility with RT-PCR. PK (600 ug/ml) wasmixed with increasing amounts of test inhibitor and allowed to react for3 min at room temperature. RNase A was added and the mixture wasincubated for 10 min. The RNase A was mixed with RNaseALERT™ substrate(AMBION®, Catalog No. AM1964) and incubated for 10 min. The fluorescenceof the RNaseALERT™ substrate was measured at 520 nm. PMSF, AAPV (SEQ IDNO:1), leupeptin, aprotinin, BSA, and antipain were found to be capableof inhibiting PK at room temperature. Under the conditions employed,TLCK, AEBSF, N-acetyl-Eglin C, benzamidine, and AAPA (SEQ ID NO:3) didnot show inhibition of PK. The AAPA (SEQ ID NO:3) preparation obtainedfrom the supplier appears to have been inferior since AAPA (SEQ ID NO:3)is expected to be inhibitory for PK.

Compatibility with RT-PCR was studied by combining each inhibitor with alysis solution (100 mM Tris pH 7.5, 25 mM MgCl₂, 5 mM CaCl₂, 0.1% TRITONX-114™, 100 U/ml DNase, 600 ug/ml PK) and purified HeLa RNA (1.8 ug).The samples were reverse transcribed using a Reverse Transcriptase Kit(Applied Biosystems Catalog No. 4368813) and the resulting cDNA wassubjected to real time PCR with the TAQMAN® Universal PCR Master Mix(Applied Biosystems Catalog No. 4304437).

Minimal RT-PCR inhibition was seen with AAPV (SEQ ID NO:1) and PMSF,each at concentrations up to 2 mM in the stopped mixture (e.g., up to400 uM in an RT reaction). Aprotinin was inhibitory at allconcentrations tested. In a similar study, these 3 inhibitors weretested in 1-step RT-PCR and only AAPV (SEQ ID NO:1) was compatible. BothPMSF and aprotinin showed RT-PCR inhibition for the one-step reaction.

These data demonstrate that AAPV (SEQ ID NO:1) and PMSF are effectiveinhibitors of proteinase K. In some embodiments, the concentration ofPMSF is kept below about 2 mM in the stopped mixture. For one-stepRT-PCR, AAPV (SEQ ID NO:1) is effective. To determine the concentrationof AAPV (SEQ ID NO:1) for inhibition of proteinase K in the lysissolution, 50 uL lysis solution was mixed with 5 uL of stop solution withvarying amounts of AAPV (SEQ ID NO:1) (11 mM Tris, 44 mM EGTA, 0.5-1 mMAAPV (SEQ ID NO:1)) and incubated for 10 min. RNase A (15 ug) was addedto each sample and the samples were held for 10 min at room temperature.The reaction mixture was heated for 30 min at 95° C. and analyzed usinga Protein 50 Bioanalyzer chip (2100 Bioanalyzer, Agilent, Santa Clara,Calif.). The Bioanalyzer protein gel data are provided by FIG. 7 inwhich the lower marker is at about 3.5 kDa; the next higher prominentbands are system peaks at about 4.0-4.5 kDa; intact RNase A is at about20 kDa; and proteinase K is at about 35 kDa. Lanes are labeled asfollows: (L) Ladder, (1) RNase Only, (2) PK Only, (3)-(8) AAPV (SEQ IDNO:1) at 1 mM (3), 0.75 mM (4), 0.5 mM (5), 0.25 mM (6), 0.125 mM (7),and 0 mM (8). AAPV (SEQ ID NO:1) was capable of inhibiting PK (100ug/ml) at concentrations as low as 0.25 mM (lane 6) and at even lowerconcentrations when incubated with PK for a longer period of time priorto addition of the RNase A substrate.

The ability of EGTA to inhibit DNase I activity and the limiting amountof EGTA that could be used in RT-PCR was determined. EGTA (88 mM; finalconc. of 8 mM) was added to the stop solution for DNase I inactivation.The pH of the stop solution was 8.0 for AAPV (SEQ ID NO:1) inactivationof proteinase K. Tris-base with HCl as needed for pH adjustment (11 mM;final conc of 1 mM) was used for buffering the solution. EGTA was foundto inhibit DNase I at 4 mM or higher.

EXAMPLE 4 Isothermal Sample Preparation Embodiments

Exemplary non-limiting embodiments of lysis solutions are prepared byobtaining stock solutions of 1M Tris-base pH 8.0, 1M MgCl₂, 1M CaCl₂, 1MDTT, proteinase K at 20 mg/ml, 20% TRITON X-114™ surfactant andnuclease-free water. Stock solutions are diluted to form a lysissolution of Tris pH 8.0, 10 mM; MgCl₂, 0.5 mM; CaCl₂, 0.5 mM; a reducingagent such as DTT, β-mercaptoethanol or dithioerythritol, 0.01 mM;protease such as proteinase K, 100 ug/ml; and TRITON X-114™ surfactant,0.1%, in nuclease free water. The pH is adjusted to pH 7.8+/−0.1 withHCl at a temperature of 19° C.-25° C. (a range of pH values is about 7.5to 8.2). The lysis solution can be stored at −20° C., at 4° C., and hasbeen found to be stable at 25° C. for one year.

A lysis mixture (termed lysis buffer in FIG. 1A) is prepared bycombining the lysis solution with a deoxyribonuclease such as DNase I ata concentration of 300 U/ml (a range of 100 U/ml-600 U/ml can be used)for those embodiments in which it is desired to remove DNA. In certainembodiments, the volume of deoxyribonuclease added is less than about 1%of the volume of the final lysis reaction. Lysis can be carried out in a50 uL volume at a pH of 7.8.

Exemplary embodiments of a stop mixture include a protease inhibitorhaving inhibitory activity for the protease of the lysis mixture; and adivalent cation chelator that, by chelating divalent cations of thelysis mixture, provides for inactivation of the deoxyribonuclease of thelysis mixture. Therefore a lysis mixture and a stop mixture are tailoredto work together.

Stock solutions for an exemplary stop mixture include a proteaseinhibitor such as AAPV (SEQ ID NO:1) in DMSO (100 mM), 1M Tris-base pH8.3, a cation chelator such as 200 mM EGTA, a reducing agent such as 1MDTT and nuclease free water. An exemplary stop mixture for use withproteinase K and DNase I includes AAPV (SEQ ID NO:1), 11 mM; 1 M Tris pH8.3, 11 mM; 200 mM EGTA, 88 mM; RNase Inhibitor such as RIP, 2.2 U/ul;and 1M DTT, 0.11 mM in nuclease free water. The pH is adjusted to8.0+/−0.1 (at 19° C.-25° C.). with HCl or KOH as needed. For thisexemplary embodiment, 5 uL of stop solution is added to 50 uL of lysismixture to form a stopped mixture.

Certain embodiments of the processes for preparing a sample for nucleicacid analysis are carried out as follows. DNase I is mixed with lysissolution and the resultant lysis mixture is stored on ice. For 1-10⁶cultured mammalian cells, cells are pelleted (˜800×g for 5 min), themedia is removed and the cells are washed with 50 uL of 1×PBS andre-pelleted. The supernatant is removed. Adhered cells in 96- or384-well plates (1 to 10⁶ cells) can also be used with this procedure.No centrifugation is required since the cells remain adhered to theplate throughout the washing procedure.

Lysis mixture (50 ul) is added to the pellet and the pellet isresuspended by pipetting. The lysis reaction is incubated for 5 minutesat room temperature (19° C.-25° C.) or for about 8 minutes for miRNAsample preparation embodiments, also at room temperature. Stop solution(5 ul) is added directly into each lysis reaction, mixed 5× bypipetting, and incubated for 2 minutes at room temperature (19° C.-25°C.). The stopped lysate is ready for downstream nucleic acid analysis,detection and/or amplification and is used within about 20 minutes forsuch a downstream procedure or is frozen for later use.

A 5-minute lysis time, a 2-minute stop time, and mixing 5× with apipette are provided for some embodiments of nucleic acid preparationmethods of the present teachings. An 8-minute lysis time, a 2-minutestop time, and mixing 5× with a pipette are provided for embodiments ofmiRNA nucleic acid preparation methods of the present teachings.Temperatures between 16° C. and 28° C. are provided for certainembodiments of isothermal preparation methods. Washing with 50 uL PBS ormedia (without fetal bovine serum) is acceptable prior to lysis.

Nucleic acid analysis, detection and/or amplification can include areverse transcription step, a real-time PCR reaction, and/or an RNAtranscription step comprising use of an RNA polymerase. The samplepreparation process provided by teachings herein provides componentsthat minimally interfere with enzymatic activity and detection methods.

The data of FIG. 8 demonstrate the linearity and efficiency of certainsample preparation processes as provided herein using the TAQMAN® GeneExpression assay for β-actin (Applied Biosystems) over 4 logs ofcellular input from 10 cells up to 100,000 cells per lysis reaction. Thedata demonstrate good linearity down to an input of as few as 10 cells.

Sample preparation processes as provided by teachings herein arecompatible with a large number of cell lines. Table 1 provides a listingof cells lines that have been tested.

TABLE 1 Cell Lines tested using Preparation Processes of EmbodimentsHerein Cell Line Growth Source Species Source Tissue HeLa adherent H.sapiens Cervical Adenocarcinoma HepG2 adherent H. sapiens LiverCarcinoma Primary adherent H. sapiens Liver Hepatocytes SK-N-AS adherentH. sapiens Brain Neuroblast SK-N-SH adherent H. sapiens Brain NeuroblastU-87 MG adherent H. sapiens Brain Glioblastoma ME-180 adherent H.sapiens Cervical Epidermoid Carcinoma A549 adherent H. sapiens LungCarcinoma Jurkat suspension H. sapiens Acute T-Cell Leukemia PC-12loosely R. norvegicus (rat) Adrenal adherent Pheochromocytoma PT-K75adherent S. scrofa (pig) Nasal Turbinate Mucosa NIH/3T3 adherent M.musculus (mouse) Embryonic Fibroblast Raji suspension H. sapiens BLymphocyte HEK-293 adherent H. sapiens Kidney COS-7 adherent C. aethiops(monkey) Kidney CHO-K1 adherent C. griseus (hamster) Ovary NCI-H460adherent H. sapiens Lung Carcinoma DU 145 adherent H. sapiens ProstateCarcinoma K562 suspension H. sapiens Bone Marrow Leukemia U-2 OSadherent H. sapiens Osteosarcoma Huh-7 adherent H. sapiens LiverCarcinoma Neuro 2A adherent M. musculus (mouse) Brain blastoma BJadherent H. sapiens Foreskin Fibroblast

In addition, C_(T) values obtained using lysates as prepared usingprocesses provided herein were found to be essentially equivalent toC_(T) values obtained with purified RNA. Stopped lysates and purifiedRNA from 5000 HeLa cells were prepared in parallel and evaluated with 78TAQMAN® Gene Expression Assays on an Applied Biosystems 7900HT Real-TimePCR Instrument. The C_(T) value obtained from the stopped lysates isplotted against the C_(T) value for the same assay using purified RNA asshown in FIG. 9A. The linear correlation coefficient is Y(stoppedlysates)=0.951×(Pure RNA)+1.05, R²=0.933.

The assays of FIG. 9A were expanded to provide data for 40,000 culturedcells (a mixture of 5 cell lines) and evaluated with 151 TAQMAN® GeneExpression Assays on an Applied Biosystems 7500 Fast Real-Time PCRInstrument. The C_(T) value obtained from the stopped lysates is plottedagainst the C_(T) value for the same assay using purified RNA as shownin FIG. 9B. The linear correlation coefficient is Y(stoppedlysates)=0.972×(Pure RNA)+0.870, R²=0.973. These data demonstratecomparable performance using the preparation methods herein as comparedto purified RNA.

Sample preparation processes of embodiments herein use fewer samplehandling and transfer steps as compared to traditional RNA purificationstrategies. As a result, there is less potential for sample loss andvariability among replicates using the isothermal processes herein overtraditional RNA purification strategies. That some of the samplepreparation processes provided herein are less variable than traditionalRNA purification strategies is demonstrated by the following study. HeLacells (5000) were either subjected to traditional RNA purification orcell lysis using isothermal preparation methods of teachings herein.Twenty-four replicates for each preparation protocol were carried out.Samples were then evaluated using a TAQMAN® Gene Expression Assay forβ-actin. Data of FIG. 10 demonstrates a greater sensitivity andreproducibility of replicate stopped lysates compared to purified RNA.

Sample preparation processes of embodiments herein are provided formicroRNA quantitation and profiling without RNA isolation. Cells (up to10⁵-10⁶) are washed in phosphate-buffered saline and lysed for 8 minutesat room temperature. DNase treatment can be performed concurrently.Lysis is terminated at room temperature for two minutes with stopsolution as described above. All of the small RNA species present in acell are available for analysis since the samples are processeddirectly. The present sample preparation embodiments have beendemonstrated to provide performance equivalent to purified RNA whentested against a panel of 111 TAQMAN® MicroRNA Assays.

Sample preparation processes of embodiments herein also provide theability to distinguish between highly homologous mature miRNA targetsfor accurate miRNA expression analysis. For example, the ability ofTAQMAN® MicroRNA Assays to distinguish between the highly homologouslet-7 family of miRNAs was not affected by the use of sample preparationprocesses described herein.

Sample preparation processes of embodiments herein also provide methodsfor analyzing effects of siRNAs for RNA interference activity. Forexample, duplicate sets of 4000 cells were transfected with 20 differentsiRNAs and the sets were either lysed using sample preparation processesof embodiments described herein or using the MAGMAX™ RNA purificationprotocol (Applied Biosystems). The percent remaining expression oftarget mRNAs was determined for each set and was found to be essentiallyequivalent. Therefore, the sample preparation embodiments providedherein provide expression data for RNA interference that is essentiallyequivalent to that of data obtained from purification of the RNA.

Sample preparation processes of embodiments herein also provide methodsfor single nucleotide polymorphism (SNP) detection. Lysates weregenerated as described herein from 10,000 HeLa cells, HepG2 cells,Jurkat cells, DU-145 cells, HEK-293 cells, K562 and MCF-7 cells in 50 μlof lysis solution and compared separately to purified DNA from the samecell sources. The TAQMAN® Genotyping Master Mix (Applied Biosystems) wasmixed with the SNP assay and 1ng of DNA (0.5 μl) or 0.5 μl of lysates(˜83 cells) was added. PCR was carried out for 40 cycles. FIG. 11provides data for detection of a SNP obtained from the two methods. “A”contains the genomic DNA and lysates from the HepG2, 293, Jurkat andK562 cell lines. “B” contains the genomic DNA and lysates from HeLacells. “C” contains the genomic DNA and lysates from MCF-7 and DU-145cell lines. The data are clustered for allele Y at “A,” for allele X at“C” and for cells containing both alleles at “B” thereby showingconsistency of results from the two methods. The “D” data provide the notemplate control samples. Therefore, sample preparation processes ofembodiments herein are equivalent in efficacy for determination of SNPdetection.

The compositions, methods, and kits of the current teachings have beendescribed broadly and generically herein. Each of the narrower speciesand sub-generic groupings falling within the generic disclosure alsoform part of the current teachings. This includes the genericdescription of the current teachings with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications can be made without departingfrom the teachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings can befurther understood in light of the following claims.

What is claimed is:
 1. A composition comprising: a polypeptide having deoxyribonuclease activity and a surfactant that substantially lacks fluorescence between 300 nm and 750 nm when in use for in situ analysis of nucleic acid, wherein the composition is substantially free of a cation chelator.
 2. The composition of claim 1 further comprising a ribonuclease inhibitor.
 3. The composition of claim 1 further comprising a reverse transcriptase.
 4. The composition of claim 1 further comprising a polymerase.
 5. The composition of claim 1 wherein the surfactant comprises octylphenol ethoxylate having an average of 7.5 ethoxylate groups.
 6. The composition of claim 1 wherein the surfactant comprises octylphenol ethoxylate having an average of 9.5 ethoxylate groups.
 7. The composition of claim 1 wherein the surfactant comprises dodecyl alcohol polyoxyethylene ether having an average of 9.5 ethoxylate groups.
 8. The composition of claim 1 wherein the surfactant comprises octylphenolpoly(ethyleneglycolether).
 9. The composition of claim 1 comprising less than 0.1 mM cation chelator.
 10. The composition of claim 1 comprising less than 0.2 mM cation chelator.
 11. The composition of claim 1 comprising less than 0.5 mM cation chelator.
 12. The composition of claim 1 comprising less than 1 mM cation chelator.
 13. A composition comprising: a polypeptide having deoxyribonuclease activity and a surfactant that substantially lacks fluorescence between 300 nm and 750 nm when in use for in situ analysis of nucleic acid, wherein the composition is substantially free of a cation chelator, wherein the surfactant comprises octylphenol ethoxylate having an average of 7.5 ethoxylate groups, and wherein the composition comprises less than 1 mM cation chelator.
 14. The composition of claim 13 comprising less than 0.5 mM cation chelator.
 15. The composition of claim 13 comprising less than 0.2 mM cation chelator.
 16. The composition of claim 13 comprising less than 0.1 mM cation chelator.
 17. A composition comprising: a polypeptide having deoxyribonuclease activity and a surfactant that substantially lacks fluorescence between 300 nm and 750 nm when in use for in situ analysis of nucleic acid, wherein the composition is substantially free of a cation chelator, wherein the surfactant comprises octylphenol ethoxylate having an average of 9.5 ethoxylate groups, and wherein the composition comprises less than 1 mM cation chelator.
 18. The composition of claim 17 comprising less than 0.5 mM cation chelator.
 19. The composition of claim 17 comprising less than 0.2 mM cation chelator.
 20. The composition of claim 17 comprising less than 0.1 mM cation chelator. 